Abstract Insulin stimulates glucose uptake in muscle by mobilizing intracellular GLUT4 storage vesicles (GSVs), which fuse at the cell surface and insert GLUT4 glucose transporters into the sarcolemma. The differential targeting of GLUT4 in basal and insulin-stimulated cells determines insulin responsiveness. Insulin resistance results from impaired GSV regulation, and contributes to the pathogenesis of the metabolic syndrome and type 2 diabetes. Defects in both insulin signaling and vesicle trafficking may contribute to impaired GSV regulation. Signaling defects have been well studied, but trafficking defects are not characterized. Recent data suggest that GLUT4 trafficking defects may be an important contributor to insulin resistance in muscle. However, even normal GSV trafficking pathways are poorly defined. This proposal builds on recent work that, for the first time, defines a pool of insulin-regulated GSVs in molecular terms. These vesicles are retained intracellularly by TUG, which links GSVs to the Golgi matrix in unstimulated cells. Insulin causes TUG cleavage to release GSVs and to insert GLUT4 at the plasma membrane. Although GSV trafficking is controlled by insulin at multiple steps, data suggest that the TUG pathway is a major site of regulation, which is compromised in diet- induced insulin resistance in mice. Moreover, GSVs contain proteins other than GLUT4, notably IRAP, which may mediate distinct physiologic actions to control vascular tone and water homeostasis. Thus, impaired GSV trafficking may result not only in insulin resistance (with respect to glucose uptake) but also contribute to other abnormal physiology. Here, we propose to test the contribution of the TUG pathway in muscle to glucose homeostasis and to other aspects of physiology. Using transgenic mice, Aim 1 will test effects of disrupting TUG action in muscle on glucose uptake and turnover, energy expenditure, and other metabolic endpoints. Aim 2 will study mice rendered insulin-resistant by a high-fat diet, and elucidate whether the trafficking and/or signaling defects that contribute to insulin resistance are bypassed by TUG disruption. Aim 3 will study how disruption of TUG action affects water homeostasis and blood pressure. It is anticipated that, together, these studies will provide fundamental new insights that are highly significant for understanding glucose homeostasis, insulin resistance, and the metabolic syndrome. Public Health Significance: Type 2 diabetes and pre-diabetes are an enormous public health burden, estimated to affect >40% of adults in the United States. These metabolic abnormalities frequently occur as part of a constellation of abnormalities, including high blood pressure, which leads to substantial morbidity and mortality. The research proposed here will investigate how these abnormalities occur, and whether distinct features of this metabolic syndrome may have a shared pathophysiologic basis.